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Azadirachtin Biosynthesis Induction in Azadirachta indica A. Juss

Cotyledonary Calli with Elicitor Agents

Article  in  Brazilian Archives of Biology and Technology · April 2014

DOI: 10.1590/S1516-89132014000200001

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 155

Vol.57, n.2: pp. 155-162, March-April 2014 BRAZILIAN ARCHIVES OF

ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L

Azadirachtin Biosynthesis Induction in Azadirachta indica A.

Juss Cotyledonary Calli with Elicitor Agents
Marcelo Rodrigues1*, Reginaldo Alves Festucci-Buselli2, Luzimar Campos Silva3 and
Wagner Campos Otoni3
1
Departamento de Biologia; Setor de Fisiologia Vegetal; Universidade Federal de Lavras; Lavras - MG - Brasil.
2
Centro de Tecnologia Agropecuária; Instituto Sócio Ambiental e dos Recursos Hídricos; Universidade Federal
Rural da Amazônia; 66077-530; Belém - PA - Brasil. 3Departamento de Biologia Vegetal; Universidade Federal de
Viçosa; Viçosa - MG - Brasil

ABSTRACT
The use of cell and plant tissues culture techniques to produce economically important active metabolites has been
growing. Among these substances, azadirachtin (AZA), produced by the neem tree (Azadirachta indica), has
received considerable attention due to its bioinsecticide action. The main goal of this work was to analyze the AZA
levels in neem cotyledonary calli. The calli were grown in agitated Woody Plant Medium (WPM) liquid medium,
supplemented with glucose (Gl), hydrolyzed casein (HC) and methyl jasmonate (MeJ) as elicitor agent. An
interaction was observed between these substances, depending on in vitro cultivation time with orbital agitation.
The highest concentrations (average of 0.2470 µg g-1) of AZA were produced in the first and second weeks of
culture when the cell mass was grown in a medium with 2% Gl v/v, 500 mg L-1 HC and 100 µM of MeJ. This
corresponded to approximately 57% of the AZA content stored in the donor plants seeds, used as a source of
explants to induce in vitro callus formation. It was concluded that the nutrition, as well as the concentration of MeJ
as signal transduction of secondary metabolism in neem cells, might influence the AZA content produced in vitro.

Key words: Neem, HPLC, glucose, hydrolysed casein, methyl jasmonate

INTRODUCTION reforestation projects, or as an ornamental tree for

The neem tree (Azadirachta indica A. Juss), syn
Antela azadirachta, or Melia azadirachta L.
belongs to the Meliaceae family (Cunha 2002). It
is originated from Asia, native to Burma and arid
regions of the Indian subcontinent, where there are
approximately 18 million trees. It is currently
grown in Australia, Africa and the Americas also.
According to the US National Council of
Investigation, neem has become one of the most
promising plants worldwide for its various
applications (Kundu 1999). It is used in human
and veterinary medicine, in the cosmetics industry,
as green manure, as a hardwood, and also for
*
Author for correspondence: marcel.or.7@hotmail.com
landscaping.
The neem tree produces more than 300 secondary
metabolites, a third of which are tetratriterpenoids
(limonoids) with different biological effects and
commercial applications (Dai et al. 2001; Neves et
al. 2003; Rodrigues et al. 2012). The best known
active compounds are azadirachtin (AZA), an
insecticide and insect repellent, nimbin, a
spermicide and anti-inflammatory in vertebrates,
and salanim, another insect repellent. Of these,
AZA (Fig. 1), which is stored mainly in the leaves
and seeds Thengane et al. (1995), is the most
economically important and abundant metabolite
produced by A. indica (Mordue and Blackwell

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
156 Rodrigues, M. et al.
1993). AZA may cause changes in the insect
development by repellent and intoxicating effects,
affecting the physiology, oviposition and egg
viability (Chopra 1958; Neves et al. 2003).
Observations of the feeding behavior of some
larvae (Spodoptera littoralis and Mamestra
brassicae) indicated that AZA might also reduce
the food consumption (Neves et al. 2003).
Figure 1 - The AZA molecule structure (C35H44O16) is a
tetratriterpenoid produced by Azadirachta
indica, mainly stored in seeds and leaves.
AZA is a complex molecule that would be difficult
to synthesize chemically. Thus, the cell and tissue
culture approaches are the best options for in vitro
production of these metabolites. The use of
biotechnology may optimize the production of
bioactive compounds through the use of elicitor
agents and other methods (Prakash et al. 2002;
Festucci-Buselli et al. 2008; Nakabayashi et al.
2010). However, AZA has not always been easy to
detect using the standard techniques. For instance,
Akula et al. (2003) was unable to detect the AZA
in somatic embryos of neem using HPLC, even
though the extract of somatic embryos was known
to affect the development of locust larvae. This
showed that somatic embryos synthesized the
AZA, or another metabolite that affected the insect
development. These apparent problems in HPLC
analysis by UV detection may be caused by the
low sensitivity of the detector, which lacks a
strong-UV absorbing molecular chromophore.
Such low sensitivity could cause errors in the
quantification of AZA (Schaaf et al. 2000).
Secondary metabolites are produced by the plants
mainly under stress conditions. The stresses may
trigger the plant defense mechanisms, leading to a
variety of biochemical signals, which induce the
expression of genes related to secondary
Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
metabolite biosynthesis. The discovery of new
compounds and their elicitors may allow the
manipulation of their biosynthetic routes,
increasing their yield in the tissue and cell cultures
(Kim et al. 2009). To increase the yield, many
scientists have used a technique based on the
application of elicitors to a cultivation medium.
This causes stress in the plant cells, signaling and
activating defense mechanisms in the plants.
Commonly used elicitors include carbohydrates,
jasmonic acid (JA), methyl jasmonate (MeJ),
salicylic acid (SA), chitosan and heavy metal ions
(Nascimento and Fett Netto 2010).
The aim of this study was to assess in vitro
production of AZA, using neem cotyledonary calli
in agitated liquid medium cultures under the
influence of different elicitor agents: glucose (Gl),
hydrolysed casein (HC) and methyl jasmonate
(MeJ).
MATERIALS AND METHODS
Callus induction
Neem plants (Azadirachta indica A. Juss)
germinated in vitro were used as explant source.
Seeds were collected at the Nim Brazil nursery
garden in São José do Rio Preto, SP, Brazil.
Cotyledons were inoculated on semisolid Woody
Plant Medium - WPM Lloyd and McCown (1981)
with 6 g L-1 granulated agar (Merck®, Germany),
and 3 mg L-1 6-benzyladenine (BA) and 0.5 mg L-1
indolyl-3-butyric acid (IBA) (adapted
methodology from Rodrigues et al. 2009), pH to
5.8. The medium was sterilized by autoclaving at
120ºC during 15 min Aliquots (10 mL) were added
to the test tubes (25 X 150 mm) and closed with
transparent polypropylene lids.
Callus cultivation
The calli were cultured in the flasks (90 mm
height, 50 mm diameter sealed with rigid
poplyproplylene lids), containing 40 mL of
semisolid WPM medium, added with 3 mg L-1 BA
and 0.5 mg L-1 IBA, and sub-cultured every 20
days. Six calli were transferred to each flask, with
the same medium, growth regulators and
concentrations, added with 200 mg L-1 myo-
inositol, 0.5 g L-1 polyvinyl pyrrolidone (PVP), 2%
sucrose v/v, and B5 vitamins Gamborg et al.
(1968) until the obtention of fresh mass calli for
setting the experiment. The medium pH was
adjusted to 5.8 previously to autoclaving at 120°C
 In vitro Production of Azadirachtin
during 15 min. The calli were cultivated in the
dark. Three samples of cotyledonary calli were
kept in 125 mL Erlenmeyer flasks with 40 mL of
WPM liquid medium, with the same growth
regulators and concentrations and placed on an
orbital shaker at 90 rpm.
Cultivation with inducing agents
The agitated WPM liquid cultures were added with
the following combinations of elicitors and
evaluated for in vitro AZA production: 1- glucose
(Gl) concentrations as carbon source (0, 1, 2,
and 4 % v/v); 2 - hydrolyzed casein (HC)
concentrations as nitrogen source (0, 250, 500,
1000 mg L-1); 3 - methyl jasmonate (MeJ)
concentrations as elicitor (0, 50, 100, 200 µM)
versus time. Three treatments were used: T0
(control); T1 (1% Gl v/v + 250 mg L-1 HC + 50
µM MeJ); T2 (2% Gl v/v + 500 mg L-1 HC + 100
µM MeJ); and T3 (4% Gl v/v + 1000 mg L-1 HC +
200 µM MeJ). Weekly periodic sampling (W1,
W2, W3 and W4) was conducted. At the end of
each week, three Erlenmeyers were sampled from
each of the four treatments (12 Erlenmeyers in
total). The dry mass was calculated after storage in
an oven at 60oC for 24 h, establishing a default
value of 0.75 g per Erlenmeyer collected. The
neem seeds were milled and dried to obtain the dry
mass, mixed with methanol, filtered and injected
into High Performance Liquid Chromatography
(HPLC) in order to compare the AZA content
produced in the treatments with natural levels.
Methanol soluble extract obtainment
Callus dry mass and seed dry mass were divided
into three 0.25 g portions and stored in an
Eppendorf with 1.0 mL of methanol per sample.
After 24 h, the samples were centrifuged at 3000
rpm for 10 min, and subsequently injected into
HPLC to determine the level of AZA present in
the methanol soluble extract.
AZA content determination
AZA content analysis was performed using the
HPLC (Shimadzu SPD-M10AVP Diode Array
Detector) equipment, following the method
described by Schaaf et al. (2000), with some
modifications. Ultraviolet (UV) was used as
detector with a 217 nm wavelength. A Bondesil
C18 reverse phase column (5 µm x 4.6 mm x 250
mm) was used, with 1.0 mL min-1 flow and
column pressure at 97 Kgf. The mobile phase
solvent was methanol and water (50:50 v/v), and
Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
157
the injected volume for each run was 50 µL. The
run time was 30 min. The mobile phase was
filtered with 0.45 µm Millipore membrane and
degassed with helium. The data were integrated by
the Shimadzu SPD software. To set the calibration
curve, AZA with 95 % purity (Sigma Aldrich,
EUA) was used at concentrations of 0; 0.0125;
0.025; 0.05; 0.1; 0.2 and 0.4 µg L-1 in methanol.
The resulting chromatogram values were plotted
and a linear equation (Fig. 2) was used to calculate
the AZA content of the samples. The natural
extract of neem seeds was analyzed under the
identical conditions.
Experimental design
A simple factorial design (3x4) was used,
comparing the concentrations of AZA and the
elicitor agents over different collection weeks,
with nine replicates per treatment. The data were
submitted to the variance analysis of the GENES
software, version Windows/2004.2.1 to Cruz
(2002), using the Tukey post-hoc test with two
factors and a 5% significance level.
RESULTS AND DISCUSSION
Azadirachtin is a tetraterpenoid and is the most
abundant molecule in neem seeds when compared
to other limonoids. The detected AZA content of
the seed extract was higher than the cultivated calli
when analyzed with a UV wavelength of 217 nm.
Such contrasts could occur when there was a
differential in vitro gene expression of several
neem secondary metabolites. Differences in the
gene expression are caused by the somaclonal
variation in the explants during the sub-cultures
(Kota et al. 2006; Lima et al. 2008). On the other
hand, this somaclonal variation can be a useful
source of new variation for genetic breeding,
mainly for the selection of cell lines with higher
yield (Carvalho et al. 2013). The a appearance of
the calli (Fig. 2A) cultivated in the semisolid
medium prior to liquid medium as well as the
AZA calibration curve is shown in Figure 2B. A
two-phase system was used for the induction of
the rapid cell growth. Growth regulators were used
and then the cells were exposed to a liquid
medium containing elicitor agents: Gl as carbon
source, HC as nitrogen source and MeJ as signal
transduction of secondary metabolism in neem
cells in vitro.
158 Rodrigues, M. et al.

Figure 2 - Azadirachta indica cotyledonary calli subcultured every 30 days in Woody Plant Medium -
WPM. A) Calli from the ninth subculture in semisolid medium, then transferred to liquid
medium; B) Linear regression of the AZA calibration, with R2 = 0.999.

The AZA production process can be optimized, when

two phases are taken. The first phase is characterized by
high nitrogen and phosphorus contents to increase the
biomass. The second phase uses low concentrations of
minerals, inducing the nutritional stress in the explants
and reducing the oxygen transference rate (OTR).
Consequently, there may be increase in the AZA
production. According Raval et al. (2003), the highest
AZA concentration (0.8 mg L-1 extract) was related
with the cells at the ninth day of cultivation in White’s
Medium (White 1963). However, after applying this
two-phase system, the highest AZA concentration Figure 3 - HPLC chromatogram. Retention time of the
produced in vitro reached 67.5% (2.7 mg/L-1 extract) in standard – 95% purity AZA (Sigma Aldrich,
callus cultures from the shoots, using the modified USA): b = 13.617 min, dotted line; treatment 2
Murashige and Skoog (MS) medium with different and week 2 (T2W2 = 2% Gl v/v + 500 mg L-1
concentrations of nitrogen and phosphorus sources in HC + 100 µM MeJ): a = 13.525 min in solid
suspension culture callus, relative to the average line.
content of the compound in natural seeds (Raval et al.
2003). These results indicated that the biomass growth
was not directly related to the in vitro production of
AZA, but directly related to the decrease of the OTR
associated with the stationary phase of cell growth. The
nutrient type and its concentration are primordial to the
cell expansion and development.
Based on the chromatogram (Fig. 3), the standard AZA
retention time was 13.617 min, with higherer
production of AZA in second week, when the cell was
cultivated in WPM liquid medium with 2% Gl v/v +
500 mg L-1 HC and 100 µM of MeJ (T2W2) took
13.525 min. By superposition, the analyzed compound
was considered AZA due to the similarity of the
retention time of the standard and sample. Figure 4 - AZA concentration (µg per gram of callus dry
There was significant interaction between the elicitor mass) as affected by elicitor agents: T1 [1% glucose (Gl)
agent concentrations as a function of sample collection v/v + 250 mg L-1 hydrolized casein (HC) + 50 µm methyl
time. The highest AZA concentration was produced by jasmonate (MeJ)]; T2 [2% (Gl) v/v + 500 mg L-1 (HC) +
the T2 treatment with 2% Gl v/v, 500 mg L-1 HC and 100 µM (MeJ)] and T3 [4% (Gl) v/v + 1000 mg L-1 (HC) +
100 µM MeJ (Fig. 4). 200 µM (MeJ)], as a function of the weekly collection (W1
to W4).
The lowercase letters indicate differences among weeks in
a same concentration of elicitors agents and the uppercase
letters indicate differences for a week among
concentrations, at 5% by the Tukey's test.

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
 In vitro Production of Azadirachtin
The first and second weeks had the highest
production rates with an average AZA
concentration of 0.247 µg per gram of callus dry
mass in the fifth subculture at 15 days intervals.
The control samples (without elicitor agents)
resulted AZA only in traces, and therefore, no area
integration was performed (in chromatograms).
Apparently increased production of bioactive
compounds with use of elicitor agents, may relate
to the “Rate Growth Hypothesis” Stamp (2003),
which suggests that when the maximum growth
decreases, the rates from the constitutive defense
levels increase. The lower amount of carbon and
nutrients assimilated by the cell – related to the
increase in osmotic potential – cause an increase in
secondary metabolites production. The AZA
content of the treatment T2W2 (2% Gl v/v + 500
mg/L-1 HC + 100 µM MeJ) corresponded to 57%
of the AZA stored in the seeds derived from the
adult neem plants (0.4301 µg g-1).
Abiotic stresses can affect the chemical
composition and amount of essential oils produced
by the plants. The biochemical and chemical
agents may favor the biochemical signaling of the
different metabolic pathways the production of
bioactive compounds (Guillon et al. 2006). The
perception of the elicitor signal leads to the
activation of transduction factors that regulate the
expression of the genes involved in the
biosynthesis of secondary metabolites. These
substances can also modify the conformation, or
activation of kinase receptors by activating their
effectors, such as ion channels, protein G, and
lipases, which are responsible for translating the
elicitor signal into the plant’s defense response.
The locations of the ligation of elicitor signals
have been identified in the plasma membrane of
various plant species. These signals can be
biochemical or chemical, and are able to
effectively influence the synthesis of secondary
metabolites in vitro (Zhao et al. 2005).
Glucose is a better carbon source than sucrose for
biomass production and is capable of stimulating
higher production of the AZA, because glucose is
a monosaccharide that has better access to the
cells, being a primary energy source to the cells.
Apparently, sucrose, after being taken up from the
culture media, was hydrolyzed to glucose and
fructose by the cell wall bound invertases
(Martinez and Park 1993). The slow consumption
of sucrose might be due to low activity of the
invertase. The selection of carbon source (glucose,
or sucrose) has long been recognized as an
Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
159
important factor in the production of secondary
metabolites by the plant cell cultures
(Chattopadhyay et al. 2002).
Nitrate nitrogen is a better nitrogen source than
ammonia for biomass production and for
stimulating AZA accumulation in vitro. The
maximum biomass was 15.02 g L-1 calli and 2.98
mg/g-1 of AZA per gram of callus, 12 days after
the cultivation of the suspension culture. Seed
kernel (along with embryo) was, thereafter,
excised and used as an explant for callus induction
on MS medium, optimizing the nutritional
composition of the culture medium and additives.
Indeed, the high ammonium concentration
inhibited the cell growth and AZA production
(Prakash and Srivastava 2005). According to
Sujanya et al. (2008), 94.41 mm of nitrate, 23.60
mm (4:1) of ammonium and a reduction in sucrose
content (15 mg L-1) increased the AZA production
(5.59 mg L-1) in MS medium with 1.0 mg L-1 NAA
and 3.0 mg L-1 BA. However, a biomass reduction
(7.4%) was also recorded when the cell
suspensions were established from the nodal
segments. Some solutes of low molecular weight
such as ammonium, aminoacids (proline) and
poliols (sorbitol and mannitol), sugars (sucrose,
glucose, fructose and threalose), are considered
osmoprotectors because they are highly soluble
and directly influence the synthesis and
accumulation of secondary metabolites in plants
(Berenguer et al. 2008; Suriyan et al. 2009).
Nutrition is known to directly influence the
content of AZA, mainly through its impact on
carbon sources (Prakash et al. 2002). MeJ and JA,
in their role as signal transduction elements in the
pathways of secondary metabolites, have been
used to induce the production of economically
important compounds. MeJ has some limitations
for long term induction of secondary metabolites
in certain crops. For example, the cells of Silybum
marianum elicited with MeJ for the production of
silimarin showed a gradual decrease in the level of
this compound throughout the successive
subcultures (Sanchez-Sampedro et al. 2009).
Ohyama and Nitsch’s (1972) culture medium
produced the highest in vitro yield of AZA
(0.0166% dry mass). Adding elicitor agents such
as JA (100 mm) and SA at 100 mm, resulted in 6-
folds (0.095%) and 9-folds (0.14%) increase,
respectively in the production of AZA in neem
root explants (Satdive et al. 2007). Biotic and
abiotic elicitor agents have been recommended as
an important strategy to optimize the in vitro
160 Rodrigues, M. et al.
generation of secondary metabolites (Savitha et al.
2006).
JA is a signaling agent for the production of
bioactive compounds in the plants, which undergo
mechanical stress by the insects (Prakash and
Srivastava 2008). Thus, AZA production and
content observed in this work were probably
influenced by the nutrition system in the culture
medium and the concentration of agents used to
stimulate its biosynthesis. Elicitors agents such as
chitosan, SA, JA, MeJ and yeast extract have been
used in neem cell suspension to promote higher
levels of the AZA in vitro production. The
combination of these agents could enable up to 5-
folds increase in AZA production (15.9 mg L-1)
due to synergism after two days of cultivation.
Using NAA and elicitor agents in a bioreactor, the
AZA concentration reached 161.1 mg L-1. The
addition of lower concentrations of SA (14 mg L-1)
resulted in an inhibitory effect, but high
concentrations (70 mg L-1) resulted in an increase
in AZA (16.8 g L-1) (Prakash and Srivastava
2008). Another stimulator of AZA production was
high concentrations of yeast extract (100 mg L-1),
which resulted in 6.5 mg g-1 of production. The
most promising combinations of elicitors used in
neem cell suspension were SA plus JA, and
chitosan plus SA at the following concentrations:
SA = 137.3 mg L-1; JA = 2.9 mg L-1 and chitosan =
16.5 mg L-1. Under these conditions, AZA content
reached 18.4 mg g-1 due to molecule synergism
after 10 days of cultivation. However, later there
was a reduction in concentration (Prakash and
Srivastava 2008). Similar results were obtained in
the present work. Overall, in vitro yield was still
low indicating that more studies to optimize the
production of AZA were required. The
relationship between the cell growth/multiplication
and in vitro AZA production was not elucidated.
This relationship was non-linear, indicating a
complex and possibly indirect relationship
between these processes. It has been reported that
more than 70% of AZA biological activity can be
lost after 30 days of storage (Allan et al. 2002).
CONCLUSIONS
High azadirachtin concentrations were obtained in
the first and second weeks of cultivation. The
highest average value was 0.2470 µg g-1 in the
second week, when the cell mass was cultivated in
Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
an orbital agitated WPM liquid medium
supplemented with 3.0 mg L-1 BA, 0.5 mg L-1
IBA, 2% Gl v/v + 500 mg L-1 HC, and 100 µM
MeJ.
ACKNOWLEDGEMENTS
The authors thank Dr. Everaldo Gonçalves de Barros
for making available laboratory facilities for HPLC
analyses, and to Eduardo Rezende Pereira for his
technical assistance. MR was recipient of a scholarship
from CNPq.
REFERENCES
Akula C, Akula A, Drew R. Somatic embryogenesis in
clonal neem, Azdirachta indica. A. Juss. and analysis
for in vitro azadirachtin production. In Vitro Cell Dev
Biol Plant. 2003; 39(3): 304-310.
Allan EJ, Eeswara JP, Jarvis AP, Mordue AJ, Morgan
ED, Stuchbury T. Induction of hairy root cultures of
Azadirachta indica A. Juss. and their production of
azadirachtin and other important insect bioactive
metabolites. Plant Cell Rep. 2002; 21(4): 374-379.
Berenguer LC, Ballesta MCM, Viguera GC, Carvajal
M. Leaf water balance mediated by aquaporins under
salt stress and associated glucosinolate synthesis in
broccoli. Plant Sci. 2008; 174(3): 321-328.
Carvalho DC, Silva ALL, Schuck MR, Purcino M,
Tanno GN, Biasi LA. Fox grape cv. Bordô (Vitis
labrusca L.) and grapevine cv. Chardonnay (Vitis
vinifera L.) cultivated in vitro under different
carbohydrates, amino acids and 6-benzylaminopurine
levels. Braz Arch Biol Technol. 2013; 56(2): 191-201.
Chattopadhyay S, Srivastava Ak, Bhojwani SS, Bisaria
VS. Production of podophyllotoxin by plant cell
cultures of Podophyllum hexandrum in bioreactor. J
Biosc Bioeng. 2002; 93(2): 215-220.
Chopra RN. The neem (Melia azadirachta L. -
Meliaceae). In: Chopra RN, editor. Indigenous drugs
of India. 2nd ed. Nova Delhi: Academic Publishers,
1958. p. 360-363.
Cruz CD. Programa Genes, release versão Windows.
2002. Editora UFV, Viçosa.
Cunha RAS. Nim indiano: a árvore milagrosa. Vetores
e Pragas. 2002; 4: 1-5.
Dai J, Yaylayan V, Vijaya A, Raghvan GS, Pare JR,
Liu Z. Multivariate calibration for the determination
of total AZRL and simple terpenoids in neem extracts
using vanillin assay. J Agr Food Chem. 2001; 49(3):
1169-1174.
 In vitro Production of Azadirachtin
Festucci-Buselli RA, Contim LAS, Barbosa LCA,
Stuart JJ, Otoni WC. Biosynthesis and potential
functions of the ecdysteroid 20-hydroxyecdysone – a
review. Botany. 2008; 86(9): 978-987.
Gamborg OL, Miller RA, Ojima K. Nutrient
requirements of suspension cultures of soybean root
cells. Exp Cell Res. 1968; 50(1): 150-158.
Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M,
Gantet P. Hairy root research: recent scenario and
exciting prospects. Curr Opin Plant Biol. 2006; 9(3):
341-346.
Kim HJ, Ono E, Morimoto K, Yamagaki T, Okazawa
A, Kobayashi A, Satake H. Metabolic engineering of
lignan biosynthesis in Forsythia sp. cell culture. Plant
Cell Physiol. 2009; 50(12): 2200-2209.
Kota S, Raghupati-Rao ND, Chary P. In vitro response
of select regions of Azadirachta indica A. Juss
(Meliaceae) as elucidated by biochemical and
molecular variations. Curr Sci India. 2006; 91(6):
770-776.
Kundu SK. The mating system and genetic significance
of polycarpy in the neem tree (Azadirachta indica).
Theor Appl Genet 1999; 99(7-8): 1216-1220.
Lima EC, Paiva R, Nogueira RC, Soares FP, Emrich
EB, Silva AAN. Callus induction in leaf segments of
Croton urucurana Baill. Ciênc Agrotec. 2008; 32(1):
17-22.
Lloyd G, McCown B. Commercially-feasible
micropropagation of Mountain laurel, Kalmia
latifolia, by use of shoot tip culture. Proc Intl Plant
Prop Soc. 1981; 30: 421-427.
Martinez BC, Park CH. Characteristics of batch
suspension cultures of preconditioned Coleus blumei
cells: sucrose effect. Biotechnol Prog. 1993; 9: 97-
100.
Mordue AJ, Blackwell A. Azadirachtin: an update. J
Insect Physiol. 1993; 39(11): 903-924.
Nakabayashi R, Yamazaki M, Saito K. A polyhedral
approach for understanding flavonoid biosynthesis in
Arabidopsis. New Biotech. 2010; 27(6): 829-836.
Nascimento NC, Fett-Neto AG. Plant secondary
metabolism and challenges in modifying its operation
an overview. In: Fett-Neto AG, editor. Plant
secondary metabolism engineering, Methods in
Molecular Biology. Dordrecht: Humana Press; 2010.
p. 1-13.
Neves BP, Oliveira IP, Nogueira JCM. Cultivo e
utilização do nim indiano (Azadirachta indica A.
Juss.). Goiânia: EMBRAPA-CNPAF-APA (Circular
Técnica). 2003; 62: 1-31.
Ohyama K, Nitsch JP. Flowering haploid plants
obtained from protoplasts of tobacco leaves. Plant
Cell Physiol. 1972; 13(2): 229-236.
Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
161
Prakash G, Bhojwani S, Srivastava AK. Production of
azadirachtin from plant tissue culture: state of the art
and future prospects. Biotechnol Biopr Eng. 2002;
7(4): 185-193.
Prakash G, Srivastava AK. Statistical media
optimization for cell growth and azadirachtin
production in Azadirachta indica (A. Juss)
suspension cultures. Process Biochem. 2005; 40(12):
3795-3800.
Prakash G, Srivastava AK. Statistical elicitor
optimization studies for the enhancement of
azadirachtin production in bioreactor Azadirachta
indica cell cultivation. Biochem Eng J. 2008; 40(2):
218-226.
Raval KN, Hellwig S, Prakash G, Ramos-Plasencia A,
Srivastava AK, Biichs J. Necessity of a two-stage
process for the production of azadirachtin-related
limonoids in suspension cultures of Azadirachta
indica. J Biosci Bioeng. 2003; 96(1): 16-22.
Rodrigues M, Paiva R, Nogueira RC, Martinotto C,
Silva Júnior JM. In vitro morphogenesis of neem
from cotyledonary-derived explants. Rev Árvore.
2009; 33(1): 21-26.
Rodrigues M, Costa THF, Festucci-Buselli RA, Silva
LC, Otoni WC. Effects of flask sealing and growth
regulators on in vitro propagation of neem
(Azadirachta indica A. Juss.). In Vitro Cell Dev Biol
Plant. 2012; 48(1): 67-72.
Sanchez-Sampedro MA, Fernández-Tárrago J, Corchete
P. Elicitation of silymarin in cell cultures of Silybum
marianum: effect of subculture and repeated addition
of methyl jasmonate. Biotechnol Lett. 2009; 31(10):
1633-1637.
Satdive RK, Fulzele DP, Eapen S. Enhanced production
of azadirachtin by hairy root cultures of Azadirachta
indica A. Juss by elicitation and media optimization.
J Biotechnol. 2007; 128(2): 281-289.
Savitha BC, Timmaraju R, Bhagya-Laksami N,
Ravishankar GA. Different biotic and abiotic elicitors
influence betalin production in hairy root cultures of
Beta vulgaris in shake flask and bioreactor. Process
Biochem. 2006; 41(1): 50-60.
Schaaf O, Jarvis AP, van der Esch SA, Giagnacovo G,
Oldham NJ. Rapid and sensitive analysis of
azadirachtin and related triterpenoids from neem
(Azadirachta indica) by high performance liquid
cromatography atmospheric pressure chemical
ionization mass spectrometry. J Chromatogr A. 2000;
886(1-2): 89-97.
Sujanya S, Devi PB, Sai I. In vitro production of
azadirachtin from cell suspension cultures of
Azadirachta indica. J Bioscience. 2008; 33(1): 113-
120.
 162 Rodrigues, M. et al.

Suriyan CU, Kirdmanee C. Proline accumulation, White PR. The cultivation of animal and plant cells, 2nd
photosynthetic abilities and growth characters of ed. Ronald Press, New York: 1963. p. 109-112.
sugarcane (Saccharum officinarum L.) plantlets in Zhao J, Davis LC, Verpoorte R. Elicitor signal
response to iso-osmotic salt and water-deficit stress. transduction leading to production of plant secondary
Agr Sci China. 2009; 8(1): 51-58. metabolites. Biotechnol Adv. 2005; 23(4): 283-333.
Stamp N. Out of the quagmire of plant defense
hypotheses. The Quart Rev Biol. 2003; 78(1): 23-55.
Thengane S, Joshi M, Mascarenhas AF. Somatic
embryogenesis in neem (Azadirachta indica). In: Jain Received: November 13, 2012;
Accepted: October16, 2013.
SM, Gupta P, Newton R (Eds.) Somatic
Embryogenesis in Woody Plants. vol II, Important
Selected Plants. Kluwer Academic Publishers,
Dordrecht: 1995. p. 357-374.

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014

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